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Pubmed Article
Deep-brain electrical microstimulation is an effective tool to explore functional characteristics of somatosensory neurons in the rat brain.
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PLoS ONE
PUBLISHED: 02-20-2015
In neurophysiology researches, peripheral stimulation is used along with recordings of neural activities to study the processing of somatosensory signals in the brain. However, limited precision of peripheral stimulation makes it difficult to activate the neuron with millisecond resolution and study its functional properties in this scale. Also, tissue/receptor damage that could occur in some experiments often limits the amount of responses that can be recorded and hence reduces data reproducibility. To overcome these limitations, electrical microstimulation (ES) of the brain could be used to directly and more precisely evoke neural responses. For this purpose, a deep-brain ES protocol for rat somatosensory relay neurons was developed in this study. Three male Wistar rats were used in the experiment. The ES was applied to the thalamic region responsive to hindpaw tactile stimulation (TS) via a theta glass microelectrode. The resulting ES-evoked cortical responses showed action potentials and thalamocortical relay latencies very similar to those evoked by TS. This result shows that the developed deep-brain ES protocol is an effective tool to bypass peripheral tissue for in vivo functional analysis of specific types of somatosensory neurons. This protocol could be readily applied in researches of nociception and other somatosensory systems to allow more extensive exploration of the neural functional networks.
Authors: Shreeya Karki, Jan Pruszak, Ole Isacson, Kai C Sonntag.
Published: 11-30-2006
ABSTRACT
ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).
26 Related JoVE Articles!
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Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Authors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky.
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
51278
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Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons
Authors: Rajeevan T. Narayanan, Hemanth Mohan, Robin Broersen, Roel de Haan, Anton W. Pieneman, Christiaan P.J. de Kock.
Institutions: VU University Amsterdam.
The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post hoc identification and morphological reconstruction.
Bioengineering, Issue 84, biocytin, juxtasomal, morphology, physiology, action potential, structure-function, histology, reconstruction, neurons, electrophysiological recordings
51359
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Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates
Authors: Alison X. Xie, Kelli Lauderdale, Thomas Murphy, Timothy L. Myers, Todd A. Fiacco.
Institutions: University of California Riverside, University of California Riverside, University of California Riverside.
Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.
Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse
51458
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Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG
Authors: Rashna D. Balsara, Sarah E. Chapman, Ian M. Sander, Deborah L. Donahue, Lucas Liepert, Francis J. Castellino, W. Matthew Leevy.
Institutions: University of Notre Dame, University of Notre Dame, University of Notre Dame, University of Notre Dame, University of Notre Dame.
Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.
Medicine, Issue 94, PET, Positron Emission Tomography, Stroke, Cerebral Ischemia, FDG, Brain template, brain atlas, VOI analysis
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Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees
Authors: Martin Fritz Brill, Maren Reuter, Wolfgang Rössler, Martin Fritz Strube-Bloss.
Institutions: University of Würzburg.
In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB. The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi channel wire electrodes is demonstrated. Pairwise differential amplification of the micro wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long term recordings over many hours up to days, which is a clear advantage compared to conventional extra and intracellular in vivo recording techniques.
Neuroscience, Issue 89, honeybee brain, olfaction, extracellular long term recordings, double recordings, differential wire electrodes, single unit, multi-unit recordings
51750
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Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents
Authors: Mikhail Kislin, Ekaterina Mugantseva, Dmitry Molotkov, Natalia Kulesskaya, Stanislav Khirug, Ilya Kirilkin, Evgeny Pryazhnikov, Julia Kolikova, Dmytro Toptunov, Mikhail Yuryev, Rashid Giniatullin, Vootele Voikar, Claudio Rivera, Heikki Rauvala, Leonard Khiroug.
Institutions: University of Helsinki, Neurotar LTD, University of Eastern Finland, University of Helsinki.
It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal’s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.
Empty Value, Issue 88, awake, in vivo two-photon microscopy, blood vessels, dendrites, dendritic spines, Ca2+ imaging, intrinsic optical imaging, patch-clamp
51869
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Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons
Authors: Jin-Young Koh, Sadahiro Iwabuchi, Zhengmin Huang, N. Charles Harata.
Institutions: University of Iowa Carver College of Medicine, University of Iowa Carver College of Medicine, EZ BioResearch LLC.
High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.
Neuroscience, Issue 95, AP2, genotyping, glial feeder layer, mouse tail, neuronal culture, nucleic-acid extraction, PCR, tattoo, torsinA
51879
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Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice
Authors: Doris Tomas, Augustinus H. Prijanto, Emma L. Burrows, Anthony J. Hannan, Malcolm K. Horne, Tim D. Aumann.
Institutions: The University of Melbourne.
Long-lasting changes in the brain or ‘brain plasticity’ underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in ‘enriched environments’ (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of ‘DA’ genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson’s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).
Neuroscience, Issue 95, Behavior, midbrain, tyrosine hydroxylase, dopamine, plasticity, substantia nigra pars compacta
52329
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Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings
Authors: Guanxiao Qi, Gabriele Radnikow, Dirk Feldmeyer.
Institutions: Research Centre Jülich, RWTH Aachen University.
The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Neuroscience, Issue 95, Patch-clamp, paired recordings, neurons, synaptic connections, gap junctions, biocytin labeling, structure-function correlations
52358
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An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System
Authors: Colin T. E. Su, Su-In Yoon, Guillaume Marcy, Eunice W. M. Chin, George J. Augustine, Eyleen L. K. Goh.
Institutions: Duke-NUS Graduate Medical School, Nanyang Technological University.
Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.
Developmental Biology, Issue 96, Neuroscience, Channelrhodopsin-2, Co-culture, Neurons, Astrocytes, induced Pluripotent Stem Cells, Neural progenitors, Differentiation, Cell culture, Cortex
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Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus
Authors: Tharakeswari Selvakumar, Kambiz N. Alavian, Travis Tierney.
Institutions: Brigham & Women's Hospital, Harvard Medical School, Imperial College London.
Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.
Neuroscience, Issue 97, anterior thalamic nucleus, deep brain stimulation, dentate gyrus, hippocampus, epilepsy, gene expression, high-frequency stimulation, quantitative RT-PCR
52457
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Quantitative Assessment of Cortical Auditory-tactile Processing in Children with Disabilities
Authors: Nathalie L. Maitre, Alexandra P. Key.
Institutions: Vanderbilt University, Vanderbilt University, Vanderbilt University.
Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology to quantitatively assess children's cortical processing of light touch, speech sounds and the multisensory processing of the 2 stimuli, without requiring active subject participation or causing children discomfort. To accomplish this we developed a dual channel, time and strength calibrated air puff stimulator that allows both tactile stimulation and sham control. We combined this with the use of event-related potential methodology to allow for high temporal resolution of signals from the primary and secondary somatosensory cortices as well as higher order processing. This methodology also allowed us to measure a multisensory response to auditory-tactile stimulation.
Behavior, Issue 83, somatosensory, event related potential, auditory-tactile, multisensory, cortical response, child
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A Simple Stimulatory Device for Evoking Point-like Tactile Stimuli: A Searchlight for LFP to Spike Transitions
Authors: Antonio G. Zippo, Sara Nencini, Gian Carlo Caramenti, Maurizio Valente, Riccardo Storchi, Gabriele E.M. Biella.
Institutions: National Research Council, National Research Council, University of Manchester.
Current neurophysiological research has the aim to develop methodologies to investigate the signal route from neuron to neuron, namely in the transitions from spikes to Local Field Potentials (LFPs) and from LFPs to spikes. LFPs have a complex dependence on spike activity and their relation is still poorly understood1. The elucidation of these signal relations would be helpful both for clinical diagnostics (e.g. stimulation paradigms for Deep Brain Stimulation) and for a deeper comprehension of neural coding strategies in normal and pathological conditions (e.g. epilepsy, Parkinson disease, chronic pain). To this aim, one has to solve technical issues related to stimulation devices, stimulation paradigms and computational analyses. Therefore, a custom-made stimulation device was developed in order to deliver stimuli well regulated in space and time that does not incur in mechanical resonance. Subsequently, as an exemplification, a set of reliable LFP-spike relationships was extracted. The performance of the device was investigated by extracellular recordings, jointly spikes and LFP responses to the applied stimuli, from the rat Primary Somatosensory cortex. Then, by means of a multi-objective optimization strategy, a predictive model for spike occurrence based on LFPs was estimated. The application of this paradigm shows that the device is adequately suited to deliver high frequency tactile stimulation, outperforming common piezoelectric actuators. As a proof of the efficacy of the device, the following results were presented: 1) the timing and reliability of LFP responses well match the spike responses, 2) LFPs are sensitive to the stimulation history and capture not only the average response but also the trial-to-trial fluctuations in the spike activity and, finally, 3) by using the LFP signal it is possible to estimate a range of predictive models that capture different aspects of the spike activity.
Neuroscience, Issue 85, LFP, spike, tactile stimulus, Multiobjective function, Neuron, somatosensory cortex
50941
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Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica
Authors: Hui Lu, Jeffrey M. McManus, Hillel J. Chiel.
Institutions: Case Western Reserve University , Case Western Reserve University , Case Western Reserve University .
In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool. This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations. To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons. We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.
Neuroscience, Issue 73, Physiology, Biomedical Engineering, Anatomy, Behavior, Neurobiology, Animal, Neurosciences, Neurophysiology, Electrophysiology, Aplysia, Aplysia californica, California sea slug, invertebrate, feeding, buccal mass, ganglia, motor neurons, neurons, extracellular stimulation and recordings, extracellular electrodes, animal model
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Propagation of Human Embryonic Stem (ES) Cells
Authors: Laurence Daheron.
Institutions: MGH - Massachusetts General Hospital.
Cellular Biology, Issue 1, ES, embryonic stem cells, tissue culture
119
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Functional Mapping with Simultaneous MEG and EEG
Authors: Hesheng Liu, Naoaki Tanaka, Steven Stufflebeam, Seppo Ahlfors, Matti Hämäläinen.
Institutions: MGH - Massachusetts General Hospital.
We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.
JoVE neuroscience, Issue 40, neuroscience, brain, MEG, EEG, functional imaging
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Simultaneous fMRI and Electrophysiology in the Rodent Brain
Authors: Wen-ju Pan, Garth Thompson, Matthew Magnuson, Waqas Majeed, Dieter Jaeger, Shella Keilholz.
Institutions: Emory University, Georgia Institute of Technology, Emory University.
To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.
Neuroscience, Issue 42, fMRI, electrophysiology, rat, BOLD, brain, resting state
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Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Authors: Brittany Baierlein, Alison L. Thurow, Harold L. Atwood, Robin L. Cooper.
Institutions: University of Kentucky, University of Toronto.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.
Neuroscience, Issue 47, Invertebrate, Crayfish, neurophysiology, muscle, anatomy, electrophysiology
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Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Authors: Aya D. Pusic, Yelena Y. Grinberg, Heidi M. Mitchell, Richard P. Kraig.
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
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Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation
Authors: Taruna Ikrar, Nicholas D. Olivas, Yulin Shi, Xiangmin Xu.
Institutions: University of California, Irvine, University of California, Irvine.
Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis. Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.
Neuroscience, Issue 56, glutamate uncaging, whole cell recording, GFP, transgenic, interneurons
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Cross-Modal Multivariate Pattern Analysis
Authors: Kaspar Meyer, Jonas T. Kaplan.
Institutions: University of Southern California.
Multivariate pattern analysis (MVPA) is an increasingly popular method of analyzing functional magnetic resonance imaging (fMRI) data1-4. Typically, the method is used to identify a subject's perceptual experience from neural activity in certain regions of the brain. For instance, it has been employed to predict the orientation of visual gratings a subject perceives from activity in early visual cortices5 or, analogously, the content of speech from activity in early auditory cortices6. Here, we present an extension of the classical MVPA paradigm, according to which perceptual stimuli are not predicted within, but across sensory systems. Specifically, the method we describe addresses the question of whether stimuli that evoke memory associations in modalities other than the one through which they are presented induce content-specific activity patterns in the sensory cortices of those other modalities. For instance, seeing a muted video clip of a glass vase shattering on the ground automatically triggers in most observers an auditory image of the associated sound; is the experience of this image in the "mind's ear" correlated with a specific neural activity pattern in early auditory cortices? Furthermore, is this activity pattern distinct from the pattern that could be observed if the subject were, instead, watching a video clip of a howling dog? In two previous studies7,8, we were able to predict sound- and touch-implying video clips based on neural activity in early auditory and somatosensory cortices, respectively. Our results are in line with a neuroarchitectural framework proposed by Damasio9,10, according to which the experience of mental images that are based on memories - such as hearing the shattering sound of a vase in the "mind's ear" upon seeing the corresponding video clip - is supported by the re-construction of content-specific neural activity patterns in early sensory cortices.
Neuroscience, Issue 57, perception, sensory, cross-modal, top-down, mental imagery, fMRI, MRI, neuroimaging, multivariate pattern analysis, MVPA
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Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals
Authors: Romain Chery, Barbara L'Heureux, Mounir Bendahmane, Rémi Renaud, Claire Martin, Frédéric Pain, Hirac Gurden.
Institutions: UMR8165 Université Paris Sud 11, Paris Diderot 7 – CNRS.
In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain. Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4. The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of ˜100μm3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet. Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.
Neuroscience, Issue 56, wide-field optical imaging, flavoproteins, hemodynamics, olfactory bulb, sensory activity, mice
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Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System
Authors: David A. Goss, Richard L. Hoffman, Brian C. Clark.
Institutions: Ohio University.
Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.
Medicine, Issue 59, neuroscience, muscle, electromyography, physiology, TMS, strength, motor control. sarcopenia, dynapenia, lumbar
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Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution
Authors: Gayathri Nattar Ranganathan, Helmut J. Koester.
Institutions: The University of Texas at Austin.
Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (>97% spike detection efficiency), has a low rate of false positive spike detection (< 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.
Neuroscience, Issue 67, functional calcium imaging, spatiotemporal patterns of activity, dithered random-access scanning
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Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity
Authors: Sheng Li.
Institutions: University of Texas Health Science Center at Houston , TIRR Memorial Hermann Hospital, TIRR Memorial Hermann Hospital.
Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. It has been extensively used in various clinical settings. Based upon recent discoveries related to the systemic effects of voluntary breathing and intrinsic physiological interactions among systems during voluntary breathing, a new EStim protocol, Breathing-controlled Electrical Stimulation (BreEStim), has been developed to augment the effects of electrical stimulation. In BreEStim, a single-pulse electrical stimulus is triggered and delivered to the target area when the airflow rate of an isolated voluntary inspiration reaches the threshold. BreEStim integrates intrinsic physiological interactions that are activated during voluntary breathing and has demonstrated excellent clinical efficacy. Two representative applications of BreEStim are reported with detailed protocols: management of post-stroke finger flexor spasticity and neuropathic pain in spinal cord injury.
Medicine, Issue 71, Neuroscience, Neurobiology, Anatomy, Physiology, Behavior, electrical stimulation, BreEStim, electrode, voluntary breathing, respiration, inspiration, pain, neuropathic pain, pain management, spasticity, stroke, spinal cord injury, brain, central nervous system, CNS, clinical, electromyogram, neuromuscular electrical stimulation
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Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue
Authors: Jaclyn M. Schwarz.
Institutions: University of Delaware.
The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.
Neuroscience, Issue 99, Fluorescence activated cell sorting, GLT-1, Thy1, CD11b, real-time PCR, gene expression
52537
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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